Mutagenesis of the active site of glucoamylase from Aspergillus awamori

The Aspergillus awamori glucoamylase gene was modified by cassette mutagenesis, and eleven mutated enzymes were expressed by Saccharomyces cerevisiae;Four mutations were constructed to change the essential Trp 120 residue in subsite 4 to Tyr, Phe, His, and Leu. All the mutations bound maltose (G2) and isomaltose (iG2) more strongly than wild-type glucoamylase. A subsite map of the Tyr 120 mutation showed significantly higher affinities for D-glucosyl residues in subsites 1 and 2, suggesting an interaction of Trp 120 with residues located there;Three carboxylic acid residues in the active site, Asp 176, Glu 179, and Glu 180, were mutated to discover their role in catalysis. The Glu-Gln 179 mutation resulted in almost complete loss of activity with little change in binding, suggesting that Gln 179 is catalytically active. The Glu-Gln 180 mutation suggests that Glu 180 provides a strong bond with [alpha]-(1 → 4)-linked D-glucosyl residues in subsite 2, but is not involved in catalysis. The Asp-Asn 176 mutation resulted in a large decrease in catalytic activity and a moderate increase in binding of G2, iG2, and maltoheptaose (G7). A subsite map of this mutation suggests that Asp 176 may interact with Trp 120 and also be catalytically active;Four residues likely to be in the active site, based on homology with related enzymes, Tyr 116, Leu 177, Trp 178, and Asn 182, were changed to Ala, His, Arg, and Ala, respectively. The Tyr-Ala 116 mutation yielded similar though less dramatic results than the Trp 120 mutations, suggesting it has a similar role in catalysis. The Leu 177, Trp 178, and Asn 182 residues were suspected of affecting the selectivity of [alpha]-(1 → 4)- against [alpha]-(1 → 6)-D-glucosidic bond hydrolysis. The Leu-His 177 mutation showed moderate decreases in binding and catalytic rates. The Trp-Arg 178 mutation showed a substantial decrease in catalytic rate and a two-fold increase in selectivity of iG2 over G2 hydrolysis. The Asn-Ala 182 mutation gave slightly lower catalytic rates with an increased binding of G2 and decreased binging of iG2. This more than doubled the selectivity of G2 over iG2 hydrolysis with little effect on the catalytic rate

Process for enzymatic hydrolysis of starch to glucose

A process for converting starch or partially hydrolyzed starch into a syrup containing dextrose includes the steps of saccharifying starch hydrolyzate in the presence of a saccharifying starch hydrolyzate in the presence of a mutated glucoamylase or related enzyme and increasing the selectivity of the enzyme for α-(1→4)-glucosidic bonds by the glucoamylase or related enzyme by including at least one mutation, the mutation substituting an amino acid of the enzyme with at least one amino acid chosen by comparison with structurally related regions of other enzymes that selectively hydrolyze only α-(1→4) glucosidic bonds. Enzymes made in accordance with the present invention are also disclosed

Rainwater Harvesting: Diversifying the Irrigation Supply of the Coker Arboretum

The University of North Carolina, Chapel Hill (UNC) is considering a variety of measures to reduce flooding at the Coker Arboretum. As underground storage has been proposed as one recommended flood control measure by Rummel, Klepper, & Kahl LLP (RK&K) and Biohabitats Inc., this report explores the design of such storage to deliver external benefits of providing the arboretum with an auxiliary water supply for irrigation as well as reducing downstream nutrient loading. Historic rainfall data from 1947-2014 was used to analyze the performance of twenty four rainwater harvesting systems of varying capacity and location. Ultimately, the most cost effective system was determined to have 76 kgal capacity capable of replacing nearly 48% of annual arboretum irrigation demand.Master of Science in Environmental Engineerin

Curcumin reduces α-synuclein induced cytotoxicity in Parkinson's disease cell model

abstract: Background Overexpression and abnormal accumulation of aggregated α-synuclein (αS) have been linked to Parkinson's disease (PD) and other synucleinopathies. αS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase αS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. Results Here we show that curcumin can alleviate αS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of αS and extracellular addition of oligomeric αS increase ROS which induces apoptosis, suggesting that aggregated αS may induce similar toxic effects whether it is generated intra- or extracellulary. Conclusions Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.The electronic version of this article is the complete one and can be found online at: http://bmcneurosci.biomedcentral.com/articles/10.1186/1471-2202-11-5

Human α4β2 Nicotinic Acetylcholine Receptor As A Novel Target Of Oligomeric α-Synuclein

Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5′-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM) analyses, we find that only large (\u3e4 nm) oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates) exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD. © 2013 Liu et al

Improved affinity selection using phage display technology and off-rate based selection

Flow systems such as a BIAcore biosensor can be very efficient tools to isolate high affinity antibody fragments from affinity matured phage display libraries. Here we show that using flow based selection, we can readily isolate a variant with a 35-fold higher affinity, especially with a 7 fold better off-rate, compared to the parent clone after only a single round of selection from a second generation affinity matured phage display library. The flow system represents a fast method to isolate affinity improved antibody fragments and can be particularly useful for isolating antibodies to antigens that have poor solubility, are toxic to the host cell, or prone to aggregation

Toxic Oligomeric Alpha-Synuclein Variants Present In Human Parkinson’S Disease Brains Are Differentially Generated In Mammalian Cell Models

Misfolding and aggregation of α-synuclein into toxic soluble oligomeric α-synuclein aggregates has been strongly correlated with the pathogenesis of Parkinson’s disease (PD). Here, we show that two different morphologically distinct oligomeric α-synuclein aggregates are present in human post-mortem PD brain tissue and are responsible for the bulk of α-synuclein induced toxicity in brain homogenates from PD samples. Two antibody fragments that selectively bind the different oligomeric α-synuclein variants block this α-synuclein induced toxicity and are useful tools to probe how various cell models replicate the α-synuclein aggregation pattern of human PD brain. Using these reagents, we show that mammalian cell type strongly influences α-synuclein aggregation, where neuronal cells best replicate the PD brain α-synuclein aggregation profile. Overexpression of α-synuclein in the different cell lines increased protein aggregation but did not alter the morphology of the oligomeric aggregates generated. Differentiation of the neuronal cells into a cholinergic-like or dopaminergic-like phenotype increased the levels of oligomeric α-synuclein where the aggregates were localized in cell neurites and cell bodies

Olivine-rich exposures at Bellicia and Arruntia craters on (4) Vesta from Dawn FC

We present an analysis of the olivine-rich exposures at Bellicia and Arruntia craters using Dawn Framing Camera (FC) color data. Our results confirm the existence of olivine-rich materials at these localities as described by Ammannito et al. (2013a) using Visual Infrared Spectrometer (VIR) data. Analyzing laboratory spectra of various Howardite-Eucrite-Diogenite meteorites, high-Ca pyroxenes, olivines and olivine-orthopyroxene mixtures, we derive three FC spectral band parameters that are indicators of olivine-rich materials. Combining the three band parameters allows us, for the first time, to reliably identify sites showing modal olivine contents >40%. The olivine-rich exposures at Bellicia and Arruntia are mapped using higher spatial resolution FC data. The exposures are located on the slopes of outer/inner crater walls, on the floor of Arruntia, in the ejecta, as well as in nearby fresh small impact craters. The spatial extent of the exposures ranges from a few hundred meters to few kilometers. The olivine-rich exposures are in accordance with both the magma ocean and the serial magmatism model (e.g., Righter and Drake 1997; Yamaguchi et al. 1997). However, it remains unsolved why the olivine-rich materials are mainly concentrated in the northern hemisphere (~36-42{\deg} N, 46-74{\deg} E) and are almost absent in the Rheasilvia basin.Comment: Accepted for publication in Meteoritics and Planetary Scienc

Delivery of Dark Material to Vesta via Carbonaceous Chondritic Impacts

NASA's Dawn spacecraft observations of asteroid (4) Vesta reveal a surface with the highest albedo and color variation of any asteroid we have observed so far. Terrains rich in low albedo dark material (DM) have been identified using Dawn Framing Camera (FC) 0.75 {\mu}m filter images in several geologic settings: associated with impact craters (in the ejecta blanket material and/or on the crater walls and rims); as flow-like deposits or rays commonly associated with topographic highs; and as dark spots (likely secondary impacts) nearby impact craters. This DM could be a relic of ancient volcanic activity or exogenic in origin. We report that the majority of the spectra of DM are similar to carbonaceous chondrite meteorites mixed with materials indigenous to Vesta. Using high-resolution seven color images we compared DM color properties (albedo, band depth) with laboratory measurements of possible analog materials. Band depth and albedo of DM are identical to those of carbonaceous chondrite xenolith-rich howardite Mt. Pratt (PRA) 04401. Laboratory mixtures of Murchison CM2 carbonaceous chondrite and basaltic eucrite Millbillillie also show band depth and albedo affinity to DM. Modeling of carbonaceous chondrite abundance in DM (1-6 vol%) is consistent with howardite meteorites. We find no evidence for large-scale volcanism (exposed dikes/pyroclastic falls) as the source of DM. Our modeling efforts using impact crater scaling laws and numerical models of ejecta reaccretion suggest the delivery and emplacement of this DM on Vesta during the formation of the ~400 km Veneneia basin by a low-velocity (<2 km/sec) carbonaceous impactor. This discovery is important because it strengthens the long-held idea that primitive bodies are the source of carbon and probably volatiles in the early Solar System.Comment: Icarus (Accepted) Pages: 58 Figures: 15 Tables: